Selection of bacterial strains useful in allergy treatment

ABSTRACT

Bacterial strains useful in prophylaxis, inhibition and/or treatment of an allergy in a mammal are selected by screening bacterial strains for capability of producing diacylglycerol kinase (DagK). A bacterial strain which is capable of producing DagK is then selected for use in prophylaxis, inhibition and/or treatment of the allergy.

This application is a 35 U.S.C. § 371 national phase entry ofInternational Application Serial No. PCT/SE2017/050455, filed May 8,2017, and which claims the benefit under 35 U.S.C. § 119 (a), of SwedishPatent Application No. 1650620-6, filed May 9, 2016, the disclosure ofeach of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present embodiments generally relate to selection of bacterialstrains, and in particular to selection of bacterial strains useful inprophylaxis, inhibition and/or treatment of allergy, and uses thereof.

BACKGROUND

Allergies, also known as allergic reactions or diseases, are a number ofconditions caused by hypersensitivity of the immune system to somethingin the environment. Allergies include, for instance, allergic rhinitis,also referred to as hay fever; food allergies; atopic dermatitis, alsoreferred to as atopic eczema; allergic asthma; and anaphylaxis. Symptomsgenerally vary depending on the type of allergy. For instance, foodallergy, which is an abnormal immune response to food, may cause signsand symptoms that range from mild to severe itchiness, swelling of thetongue, vomiting, diarrhea, hives, trouble breathing, or low bloodpressure. This typically occurs within minutes to several hours ofexposure. When the symptoms are severe it is known as anaphylaxis.

Common allergens, i.e., antigens that produce an abnormally vigorousimmune response in which the immune system fights off a perceived threatthat would otherwise be harmless to the body, include, among others,pollen and food. The underlying mechanism involves immunoglobulin Eantibodies (IgE) binding to an allergen and then to a receptor on mastcells or basophils where it triggers the release of inflammatorychemicals, such as histamine.

Current treatments for allergies include avoiding known allergens andthe use of medications, such as antihistamines. An antihistamine is amedicament that opposes the activity of histamine receptors in the body.Antihistamines are subclassified according to the histamine receptorthat they act upon. The two largest classes of antihistamines areH1-antihistamines and H2-antihistamines. Antihistamines that target thehistamine H1-receptor (H1R) are mainly used to treat allergic reactions.Antihistamines that target the histamine H2-receptor (H2R) are used totreat conditions of the gastrointestinal system.

Some older types of antihistamine drugs are marred by side effects, suchas drowsiness and reduced coordination. Also newer types ofantihistamine drugs can have unwanted side effects, such as dry mouthand headache. Hence, there is room for further improvement of new waysto treat and prevent allergies.

Various locations of the body of humans and other mammals are inhabitedby many different species of bacteria, including a number of differentspecies of lactic acid bacteria. Such bacteria many times coexist withtheir host giving synergistic beneficial effects of various kinds,nowadays also known to be diverse and dependent upon the actual strainof bacteria. According to the currently adopted definition by Food andAgriculture Organization in the U.S. (FAO) and World Health Organization(WHO), probiotics are “live microorganisms which when administered inadequate amounts confer a health benefit on the host”. Nowadays, anumber of different bacteria are used as probiotics, for example lacticacid producing bacteria, such as strains of Lactobacillus andBifidobacteria.

One example of a known lactic acid bacterium is Lactobacillus reuteri,which is a commensal intestinal Firmicute, and probiotic that is widelyprevalent in the gastrointestinal tracts of diverse avian and mammalianspecies. This organism is considered to be generally recognized as safe(GRAS) and beneficial microbe, and has been used globally as a probioticfor approximately two decades. L. reuteri has been reported to suppresspro-inflammatory cytokines in intestinal epithelial cells, monocytes,and intestinal inflammation in different rodent models.

Lee et al. (2004) disclose that lactic acid bacteria can be used as oralallergy-therapeutic means via promoting Th1 cell cytokines, such asIL-2, while suppressing Th2 cell cytokines, such as IL-4 and IL-5.

SUMMARY

It is a general objective to select bacterial strains useful inprophylaxis, inhibition and/or treatment of allergy.

An aspect of the embodiments relates to a method for selecting abacterial strain for use in prophylaxis, inhibition and/or treatment ofan allergy in a mammal. The method comprises screening bacterial strainsfor capability of producing diacylglycerol kinase (DagK). The methodalso comprises selecting a bacterial strain which is capable ofproducing DagK for use in prophylaxis, inhibition and/or treatment of anallergy in a mammal.

Another aspect of the embodiments relates to a bacterial strain capableof producing DagK for use in prophylaxis, inhibition and/or treatment ofan allergy in a mammal with the proviso that the bacterial strain is notselected from a group consisting of Lactobacillus reuteri strain ATCCPTA-6475 and L. reuteri strain ATCC PTA-4659

A further aspect of the embodiments relates to a method of prophylaxis,inhibition and/or treatment of an allergy in a mammal. The methodcomprises administering a bacterial strain capable of producing DagK toa mammal suffering from or having a risk of developing an allergy withthe proviso that the bacterial strain is not selected from a groupconsisting of L. reuteri strain ATCC PTA-6475 and L. reuteri strain ATCCPTA-4659.

Additional aspects of the embodiments include L. reuteri DSM 32273 foruse in prophylaxis, inhibition and/or treatment of an allergy in amammal, L. reuteri ATCC PTA-6475 for use in prophylaxis, inhibitionand/or treatment of a food allergy in a mammal, L. reuteri ATCC PTA-4659for use in prophylaxis, inhibition and/or treatment of a food allergy ina mammal and L. fermentum ATCC 14931 for use in prophylaxis, inhibitionand/or treatment of an allergy in a mammal.

Another aspect of the embodiments relates to an anti-allergy compositioncomprising a bacterial strain capable of producing DagK and aH1-antihistamine.

Further aspects relate to an anti-allergy composition according to abovefor use as a medicament and for use in prophylaxis, inhibition and/ortreatment of an allergy in a mammal.

The DagK producing bacterial strains of the embodiments are capable ofterminating the diacylglycerol (DAG) signaling in the H1R signalingpathway. Accordingly, histamine released in an allergic reaction isinhibited to induce its pro-inflammatory effect via the H1R signalingpathway but is still capable of inducing its anti-inflammatory effectvia the H2R signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof,may best be understood by making reference to the following descriptiontaken together with the accompanying drawings, in which:

FIG. 1 is a flow chart illustrating a method for selecting a bacterialstrain for use in prophylaxis, inhibition and/or treatment of an allergyin a mammal according to an embodiment.

FIG. 2 illustrates that L. reuteri wild-type (WT) and hdcA mutant ATCCPTA-6475 and ATCC PTA-4659 produce DagK. Relative mRNA target geneexpression levels normalized to the house keeping gene rpoB arepresented from 3, 6, 24 and 48 hours culture of each bacterium. mRNAobtained from 3 hours culture of each bacterium were set to 1.0 and usedas calibrator to identify the relative mRNA fold difference.

FIG. 3 illustrates L. reuteri DagK amino acid sequence (SEQ ID NO: 13).The amino acid sequence of L. reuteri DagK is shown together withtrypsin cleavage sites (vertical lines). The bold amino acids indicatepeptide sequences obtained following such trypsin treatment. The blackbars indicate peptide sequences found in LC-MS/MS experiments.

FIG. 4 illustrates L. reuteri DagK amino acid sequence (SEQ ID NO: 13).The bold amino acids indicate peptide sequences obtained. The black barsindicate peptide sequences found in LC-MS/MS experiments.

FIG. 5 illustrates that a commercially available Dag K inhibitor (DGKinhi) prevents L. reuteri derived-DagK mediated protein kinase C (PKC)phosphorylation in mouse enteroids. (a) illustrates a Western blotanalysis showing proteins isolated from mouse ileal enteroids of 10week-old germ free (GF) male mice treated with only media control(LDM-4), WI Lr (WT Lr grown in LDM4), DagK inhibitor and DagK inhibitorwith WT Lr for 45 min targeting mammalian phosphorylated PKC (pPKC). 10μg proteins were loaded into each well of an SDS gel. The pPKC synthesisratio was obtained by image-J analysis where pPKC of DagK inhibitortreated group was set to 1 and used as baseline. (b) Mean values of n=3showing pPKC protein concentration (loaded 10 μg) and quantified bydensitometry using image-J. GF control groups were set to 1. *P<0.05,**P<0.01, ***P<0.001. n=10 mice per group. One-Way analysis of variancewith Bonferroni correction.

DETAILED DESCRIPTION

The present embodiments generally relate to selection of bacterialstrains, and in particular to selection of bacterial strains useful inprophylaxis, inhibition and/or treatment of allergy, and uses thereof.

The present embodiments have taken a radically different approach in thefield of allergy prophylaxis and treatment as compared to thetraditional antihistamine approach. In clear contrast to the prior art,the present embodiments are based on selecting and using bacterialstrains that are beneficial within prophylaxis, inhibition and/ortreatment of allergies. The bacterial strains selected and usedaccording to the embodiments are capable of producing diacylglycerolkinase (DagK).

DagK is an enzyme that catalyzes the conversion of diacylglycerol (DAG)to phosphatidic acid (PA) utilizing adenosine triphosphate (ATP) as asource of the phosphate. In non-stimulated cells, DagK activity is lowallowing DAG to be used for glycerophospholipid biosynthesis. However,on receptor activation of the phosphoinositide pathway, DagK activityincreases driving the conversion of DAG to PA. Conversion of DAG to PAdepletes DAG, which otherwise may activate protein kinase C (PKC).

Histamine H1-receptor (H1R) downstream signaling is interrupted by DagKsynthesis by inhibiting lipid DAG involved in the signaling.Accordingly, DagK-producing bacterial strains as disclosed hereinsuppress pro-inflammatory effects of released histamine. This in turnallows only histamine H2-receptor (H2R) activation by the histamineproduced as a result of an allergic reaction. Such H2R activationpromotes anti-inflammatory symptoms.

Thus, a bacterial strain capable of producing DagK causes a suppressionof H1R downstream signaling but on the other hand, the histaminereleased by the allergic reaction induces H2R activation, which incombination suppresses the pro-inflammatory effects of histamine andpromotes anti-inflammatory and anti-allergic symptoms.

In bacteria, the enzyme DagK is expressed by the gene dagK.

FIG. 1 is a flow chart illustrating a method for selecting a bacterialstrain for use in prophylaxis, inhibition and/or treatment of allergy ina mammal. The method comprises screening, in step S1, bacterial strainsfor capability of producing DagK. The method also comprises selecting,in step S2, a bacterial strain which is capable of producing DagK foruse in prophylaxis, inhibition and/or treatment of an allergy in amammal.

The method as shown in FIG. 1 can thereby be used to identify and selectbacterial strains that are useful to prevent, inhibit and/or treatallergy in mammals. The selection criterion is the capability ofproducing the enzyme DagK, which, as mentioned above, suppresses thepro-inflammatory effects of histamine released as a result of anallergic reaction and promotes the anti-inflammatory effects of thereleased histamine.

In an embodiment, step S2 in FIG. 1 comprises selecting a bacterialstrain which is capable of producing DagK and capable of extracellularlyreleasing the DagK. Hence, in this embodiment, the bacterial strainselected in step S2 is not only capable of producing DagK but also makessoluble DagK, which is also available extracellularly, i.e., outside ofthe cells of the selected bacterial strain. This means that thebacterial strains are capable of secreting or otherwise extracellularlyreleasing DagK.

In an embodiment, screening bacterial strains for capability ofproducing DagK is assessed by detecting presence of a gene encodingdiacylglycerol kinase, such as the dagK gene, in the bacterial strain,either in the genome thereof or in an expression cassette, such as in aplasmid. In such an embodiment, step S1 of FIG. 1 comprises screeningthe bacterial strains for presence of a gene encoding DagK, such as thedagK gene. Step S2 comprises, in this embodiment, selecting a bacterialstrain comprising the gene encoding DagK, such as the dagK gene.

In a particular embodiment, the bacterial strain comprises an activegene encoding DagK. Active with regard to the gene implies that the geneencoding DagK is controlled by a constitutive promoter in the bacterialstrain or by an inducible promoter in the bacterial strain, i.e., apromoter that may be activated or induced in the bacterial strain.

There are various techniques available that could be used to detectpresence of a gene encoding DagK. Non-limiting, but illustrativetechniques, involve polymerase chain reaction (PCR) using primerscomplementary to portions of the gene encoding DagK or complementary tothe promoter of the gene encoding DagK to amplify and detect presence ofthe amplified deoxyribonucleic acid (DNA) sequence corresponding to thegene encoding DagK, a portion thereof, the promoter of the gene, or aportion thereof. In particular, quantitative polymerase chain reaction(qPCR) can be used to detect presence of a gene encoding DagK. Othertechniques involve various DNA sequencing techniques.

In another embodiment, screening bacterial strains for capability ofproducing DagK is assessed by detecting presence of the DagK enzyme,such as in the cytosol of the bacteria cells or, if the bacterial strainadditionally is capable of secreting or extracellularly releasing DagK,in the culture medium, in which the bacterial strain is cultured. Inthis embodiment, step S1 of FIG. 1 comprises screening the bacterialstrains for presence of DagK in a cytosol of the bacterial strainsand/or presence of DagK in a respective culture medium in which thebacterial strains are cultured. Step S2 comprises, in this embodiment,selecting a bacterial strain i) comprising DagK in its cytosol and/orii) for which a culture medium in which the bacterial strain is culturedcomprises DagK.

There are various techniques that could be used to detect presence ofDagK in the cytosol and/or culture medium of the bacteria cells.Non-limiting, but illustrative, techniques include using anti-DagKantibodies, such as in an enzyme-linked immunosorbent assay (ELISA);protein mass spectrometry, such as peptide mass fingerprinting bymatrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)and electrospray ionization (ESI) TOF complemented by tandem massspectrometry (MS/MS) analysis; immunoelectrophoresis; etc.

In an embodiment, the bacterial strains screened and selected in themethod of FIG. 1 are bacterial strains generally recognized as safe(GRAS). Accordingly, the bacterial strains should preferably not causeany diseases or deleterious conditions when administered to the mammalin order to prevent, inhibit and/or treat allergy. Hence, the bacterialstrains selected according to the method in FIG. 1 are preferablynon-pathogenic bacterial strains. Such GRAS bacterial strains aretherefore preferably so-called beneficial microbes. A particular exampleof bacterial strains that are GRAS are probiotic bacteria, such asnon-pathogenic probiotic bacteria. Hence, in an embodiment, step S1 inFIG. 1 comprises screening probiotic bacterial strains for capability ofproducing DagK and step S2 comprises selecting a probiotic bacterialstrains which is capable of producing DagK.

In an embodiment, the bacterial strains are lactic acid bacterialstrains. In such an embodiment, step S1 comprises screening lactic acidbacterial strains for capability of producing DagK. Correspondingly,step S2 comprises selecting a lactic acid bacterial strains which iscapable of producing DagK.

Lactic acid bacteria, also referred to as lactobacillales, are a cladeof Gram-positive, low-GC, acid-tolerant, generally nonsporulating,nonrespiring, either rod- or cocci-shaped bacteria that share commonmetabolic and physiological characteristics. These bacteria producelactic acid as the major metabolic end product of carbohydratefermentation. Genera that comprise the lactic acid bacteria includeLactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus.Lactic acid bacteria can also be found in other genera, such as,Aerococcus, Carnobacterium, Enterococcus, Oenococcus,Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella.

Particular preferred bacterial species among Streptococcus includeStreptococcus salivarius and Streptococcus termophilus.

In a particular embodiment, the bacterial strains are S. salivarius orS. termophilus strains. In this embodiment, step S1 of FIG. 1 comprisesscreening S. salivarius or S. termophilus strains for capability ofproducing DagK and step S2, correspondingly, comprises selecting a S.salivarius or S. termophilus strain which is capable of producing DagK.

In a particular embodiment, step S2 comprises selecting a S. salivariusor S. termophilus strain which is capable of producing DagK for use inprophylaxis, inhibition and/or treatment of a pollen allergy in amammal.

A currently preferred genus is Lactobacillus.

Lactobacillus include several species including L. acetotolerans, L.acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L.alimentarius, L. amylolyticus, L. amylophilus, L. amylotrophicus, L.amylovorus, L. animalis, L. antri, L. apodemi, L. aviaries, L.bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L.catenaformis, L. ceti, L. coleohominis, L. collinoides, L. cornposti, L.concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus, L.delbrueckii subsp. bulgaricus, L. delbrueckii subsp. elbrueckii, L.delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L.equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis,L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri,L. gastricus, L. ghanensis, L. graminis, L. hammesii, L. hamster, L.harbinensis, L. hayakitensis, L. helveticus, L. hilgardii, L.homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L.johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchi, L.kitasatonis, L. kunkeei, L. leichmannii, L. lindneri, L. malefermentans,L. mall, L. manihotivorans, L. mindensis, L. mucosae, L. murinus, L.nagelii, L. namurensis, L. nantensis, L. oligofermentans, L. oris, L.panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei, L.paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius,L. paraplantarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L.protectus, L. psittaci, L. rennini, L. reuteri, L. rhamnosus, L. rimae,L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L.salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L.sharpeae, L. siliginis, L. spicheri, L. suebicus, L. thailandensis, L.ultunensis, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. vini,L. vitulinus, L. zeae, and L. zymae. In an embodiment, the bacterialstrains are lactic acid bacterial strains having GRAS status, beingnon-pathogenic and selected among the above presented group ofLactobacillus species.

In a particular embodiment, the bacterial strains are L. reuteristrains. In this embodiment, step S1 of FIG. 1 comprises screening L.reuteri strains for capability of producing DagK and step S2,correspondingly, comprises selecting a L. reuteri strain which iscapable of producing DagK.

In a particular embodiment, step S2 comprises selecting a L. reuteristrain which is capable of producing DagK for use in prophylaxis,inhibition and/or treatment of a food allergy in a mammal.

In another particular embodiment, the bacterial strains are L. fermentumstrains. In this embodiment, step S1 of FIG. 1 comprises screening L.fermentum strains for capability of producing DagK and step S2,correspondingly, comprises selecting a L. fermentum strain which iscapable of producing DagK.

In a particular embodiment, step S2 comprises selecting a L. fermentumstrain which is capable of producing DagK for use in prophylaxis,inhibition and/or treatment of a pollen allergy in a mammal.

In an embodiment, the bacterial strains are grown in presence of DAG inorder to achieve pre-activated bacteria strains already expressing DagKbefore freeze-drying or lyophilizing of the bacteria. Hence, in thisembodiment the method further comprises culturing the bacterial strainsin presence of DAG.

Another aspect of the embodiments relates to a bacterial strain capableof producing DagK for use in prophylaxis, inhibition and/or treatment ofan allergy in a mammal with the proviso that the bacterial strain is notselected from a group consisting of L. reuteri strain ATCC PTA-6475 andL. reuteri strain ATCC PTA-4659.

In an embodiment, the allergy is selected from a group consisting of afood allergy and a pollen allergy. In a particular embodiment, theallergy is a food allergy and preferably a food allergy selected from agroup consisting of peanut allergy, milk (lactose or milk protein)allergy, egg allergy, tree nuts allergy, fish allergy, shellfishallergy, soy allergy, and wheat (gluten) allergy.

Non-limiting examples of pollen allergies include allergy against pollenfrom pine (Pinus), birch (Betula), alder (Alnus), cedar, hazel(Corylus), hornbeam (Carpinus), horse chestnut (Aesculus), willow(Salix), poplar (Populus), plane (Platanus), linden/lime (Tilia), olive(Olea), sugi (Cryptomefia japonica), hinoki (Chamaecyparis obtusa),ryegrass (Lolium sp.), timothy (Phleum pratense), ragweed (Ambrosia),plantain (Plantago), nettle/parietaria (Urticaceae), mugwort (ArtemisiaVulgaris), Fat hen (Chenopodium), and sorrel/dock (Rumex).

The DagK producing bacterial strains of the embodiments can be used totreat allergy in the mammal. Treat as used herein does not necessarilyimply that the mammal becomes 100% symptom free following administrationof the bacterial strains of the embodiments to the mammal. Treat alsoencompasses reducing the symptoms of the allergy in the mammal. Hence,the bacterial strains of the embodiments can be used to inhibit orsuppress allergy in the mammal.

The bacterial strains of the embodiments could also, or alternatively,be used in prophylaxis, i.e., preventing or at least reducing the riskof a mammal to develop allergy. The mammal could, for instance, have apredisposition to allergy, such as a genetic or heredity predispositionto allergy. The bacterial strains of the embodiments could then beadministered to such a mammal to prevent or at least reduce the risk ofthe mammal suffering from allergy or developing an allergic reaction.

In a particular embodiment, the mammal to which the bacterial strains ofthe embodiments can be administered is preferably a human. The bacterialstrains of the embodiments could also be used in veterinaryapplications, i.e., administered to non-human mammals. Non-limitedexamples of such non-human mammals include dog, cat, horse and cow.

In an embodiment, the bacterial strain is a bacterial strain capable ofproducing DagK and capable of extracellularly releasing, such assecreting, DagK.

In an embodiment, the bacterial strain is a lactic acid bacterial straincapable of producing DagK. In a particular embodiment, the lactic acidbacterial strain is a lactic acid bacterial strain capable of producingDagK selected from a group consisting of Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium,Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus,Vagococcus, and Weissella. In an embodiment, the lactic bacterial straincapable of producing from DagK is selected from a group consisting ofLactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus.

In an embodiment, the bacterial strain is a L. reuteri strain capable ofproducing DagK. In a particular embodiment, the L. reuteri straincapable of producing DagK is for use in prophylaxis, inhibition and/ortreatment of a food allergy in a mammal.

In another embodiment, the bacterial strain is a L. fermentum straincapable of producing DagK. In a particular embodiment, the L. fermentumstrain capable of producing DagK is for use in prophylaxis, inhibitionand/or treatment of a pollen allergy in a mammal.

In yet another embodiment, the bacterial strain is a S. salivarius or S.termophilus strain capable of producing DagK. In a particularembodiment, the S. salivarius or S. termophilus strain capable ofproducing DagK is for use in prophylaxis, inhibition and/or treatment ofa pollen allergy in a mammal.

Yet another aspect of the embodiments relates to use of a bacterialstrain capable of producing DagK in the manufacture of a medicament forprophylaxis, inhibition and/or treatment of an allergy in a mammal, withthe proviso that the bacterial strain is not selected from a groupconsisting of L. reuteri strain ATCC PTA-6475 and L. reuteri strain ATCCPTA-4659.

A further aspect of the embodiments relates to a method of prophylaxis,inhibition and/or treatment of an allergy in a mammal. The methodcomprises administering a bacterial strain capable of producing DagK toa mammal suffering from or having a risk of developing an allergy, withthe proviso that the bacterial strain is not selected from a groupconsisting of L. reuteri strain ATCC PTA-6475 and L. reuteri strain ATCCPTA-4659.

An appropriate mode of administration and formulation of the bacterialstrains is chosen depending on the site where local production of DagKis desired. A preferred mode of administration is oral. Other modes ofadministration include nasal, intraocular, topical or some other form oflocal administration to the skin, rectum, nose, eyes, vagina or gums, orintravenous, subcutaneous or intramuscular injection.

Oral administration of the bacterial strains of the embodiments may beparticularly preferred to prevent, inhibit and/or treat food allergy ina mammal. In such a case, the bacterial strains will, due to the mode ofadministration, be close the intestinal epithelium and can thereby, bythe production of DagK by the bacterial strains, modify the lipidsignaling to cause changes in the immune biomarkers to have a localimpact on the inflammatory or allergic responses.

Nasal administration of the bacterial strains of the embodiments may beparticularly preferred to prevent, inhibit and/or treat pollen allergyin a mammal. In such a case, the bacterial strains will, due to the modeof administration, be close the respiratory epithelium and can thereby,by the production of DagK by the bacterial strains, modify the lipidsignaling to cause changes in the immune biomarkers to have a localimpact on the inflammatory responses or allergic responses.

Appropriate doses of the strains as defined herein can readily be chosendepending on the allergy to be treated, the mode of administration andthe formulation concerned. For example, a dosage and administrationregime is chosen such that the bacteria administered to the subject inaccordance with the present invention can result in desired therapeuticeffects, prophylactic effects or health benefits. Thus, preferably thedosage is a therapeutically or prophylactically effective dosage, whichis appropriate for the type of mammal and allergy being treated. Forexample, daily doses of 10⁴ to 10¹⁰, for example 10⁵ to 10⁹, or 10⁶ to10⁸, or 10⁸ to 10¹⁰ total CFUs of bacteria may be used. A preferreddaily dose is around 10⁸ total CFUs, e.g., 10⁷ to 10⁹ or 10⁸ to 10⁹.

Allergies are generally correlated with an increased histamine releasewith pro-inflammatory symptoms. Thus, DagK producing bacterial strainsof the embodiments could be a great therapeutic approach to suppressinflammation caused by the histamine released during the allergicreaction in a more natural way. This means that the bacterial strains ofthe embodiments capable of producing DagK can avoid side effects causedby antihistamine drugs.

A prophylactic or therapeutic anti-allergy effect can be achieved by thebacterial strains of the embodiments alone, i.e., as the soleanti-allergy active agent. However, the bacterial strains of theembodiments could be combined with other anti-allergy drugs, such asantihistamines, to form a composition comprising the bacterial strainsand at least one antihistamine, optionally together with apharmaceutically acceptable carrier, diluent, excipient or solvent. Acombined treatment can also be achieved by separately administering thebacterial strains of the embodiments and at least one antihistamine tothe mammal suffering from or running a risk of suffering from allergy.

An advantage of a combined treatment, in which a DagK producingbacterial strain is combined with at least one antihistamine, is thatthe required dose of the at least one antihistamine can be reduced ascompared to solely administering the at least one antihistamine. Such areduction in the antihistamine dose can thereby avoid or at leastminimize any side effects associated with the at least one antihistaminewhile still achieving sufficient anti-allergy effect.

Accordingly, in an embodiment, an amount of the at least oneantihistamine in the composition is preferably less than 90%, such as byweight, as compared to an amount of the at least one antihistamine in acomposition comprising the at least one antihistamine as the soleanti-allergy agent(s), i.e., lacking any bacterial strain of theembodiments. In various embodiments, the amount of the at least oneantihistamine in the composition is preferably less than 80%, less than70%, less than 60%, less than 50%, less than 40%, less than 30%, lessthan 20%, or less than 10%, such as by weight, as compared to an amountof the at least one antihistamine in a composition comprising the atleast one antihistamine as the sole anti-allergy agent(s), i.e., lackingany bacterial strain of the embodiments.

Non-limiting, but illustrative, examples of antihistamines that can beused together with a DagK producing bacterial strain in a combinedtreatment include H1-antishistamines, such as H1R antagonists and/or H1Rinverse agonists.

Illustrative examples of H1R antagonists include acrivastine,azelastine, bilastine, bromodiphenhydramine, brompheniramine, buclizine,carbinoxamine, cetirizine (for instance sold under the name ZYRLEX®,VIALERG®, ACURA®), chlorodiphenhydramine, chlorphenamine,chlorpromazine, clemastine, cyclizine, cyproheptadine,dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene,diphenhydramine, doxylamine, ebastine (for instance sold under the nameKESTINE®), embramine, fexofenadine (for instance sold under the nameALLEGRA®, ALTIFE®), hydroxyzine, loratadine (for instance sold under thename CLARITYN®), meclizine, mirtazapine, olopatadine, orphenadrine,phenindamine, pheniramine, phenyltoloxamine, promethazine, quetiapine,rupatadine, tripelennamine and triprolidine.

Illustrative examples of H1R inverse agonists include cetirizine,levocetirizine, desloratadine (for instance sold under the nameFLYNISE®) and pyrilamine.

Accordingly, an aspect of the embodiments relates to an anti-allergycomposition comprising a bacterial strain capable of producing DagK anda H1-antihistamine.

In an embodiment, the H1-antihistamine is selected from a groupconsisting of cetirizine, ebastine, fexofenadine, loratadine anddesloratadine.

In an embodiment, the bacterial strain is not selected from a groupconsisting of L. reuteri strain ATCC PTA-6475 and L. reuteri strain ATCCPTA-4659.

Further aspects of the embodiments relates to an anti-allergycomposition for use as a medicament and for use in prophylaxis,inhibition and/or treatment of an allergy in a mammal.

Appropriate doses of the strains as defined herein in a combinedtreatment can readily be chosen depending on the allergy to be treated,the mode of administration, the formulation concerned and the at leastone antihistamine that used in the combined treatment. For example, adosage and administration regime is chosen such that the bacteria andantihistamine(s) administered to the subject in accordance with thepresent invention can result in desired therapeutic effects,prophylactic effects or health benefits. Thus, preferably the dosage isa therapeutically or prophylactically effective dosage, which isappropriate for the type of mammal and allergy being treated. The dailydoses for the bacteria mentioned in the foregoing can be used be used inthe combined treatment. It is also possible to use lower daily doses forthe combined treatment since the bacteria are combined with at least oneantihistamine.

The bacterial strains can also be used as an adjuvant in oralimmunotherapy for the treatment of food allergy, such as peanut allergy.

Experimental data as presented herein show that L. reuteri DSM 32273(deposited under the Budapest Treaty at the DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (Inhoffenstrasse 7B, D—38124Braunschweig, Germany) on Mar. 8, 2016), L. reuteri ATCC PTA-6475(deposited under the Budapest Treaty at the American Type CultureCollection (10801 University Blvd, Manassas, Va. 20110-2209, U.S.) onDec. 21, 2004) and L. reuteri ATCC PTA-4659 (deposited under theBudapest Treaty at the American Type Culture Collection (10801University Blvd, Manassas, Va. 20110-2209, U.S.) on Sep. 11, 2002) areL. reuteri strains capable of producing DagK. Accordingly, these L.reuteri strains can be used in prophylaxis, inhibition and/or treatmentof an allergy in a mammal as disclosed herein. In a particularembodiment, the allergy is a food allergy.

Thus, an aspect of the embodiments relates to a L. reuteri strain DSM32273 for use in prophylaxis, inhibition and/or treatment of an allergyin a mammal. In a particular embodiment, the allergy is a food allergy.

Other aspects of the embodiments relates to a L. reuteri strain ATCCPTA-6475 and/or a L. reuteri strain ATCC PTA-4659 for use inprophylaxis, inhibition and/or treatment of a food allergy in a mammal.

The L. reuteri strains DSM 32273, ATCC PTA-6475 and ATCC PTA-4659 couldbe used separately or as a composition of at least two of the L. reuteristrains, such as L. reuteri DSM 32273 and ATCC PTA-6475, L. reuteri DSM32273 and ATCC PTA-4659, L. reuteri ATCC PTA-4659 and ATCC PTA-6475, orL. reuteri DSM 32273, ATCC PTA-6475 and ATCC PTA-4659.

In an embodiment, L. fermentum ATCC 14931 is a L. fermentum straincapable of producing DagK. Accordingly, L. fermentum ATCC 14931 can beused in prophylaxis, inhibition and/or treatment of an allergy in amammal as disclosed herein. In a particular embodiment, the allergy is apollen allergy.

The L. fermentum strain ATCC 14931 could be used separately or as acomposition of at least one of the above listed DagK-producing L.reuteri strains. Such a composition could, then, include L. fermentumATCC 14931 and L. reuteri DSM 32273, L. fermentum ATCC 14931 and L.reuteri ATCC PTA-6475, L. fermentum ATCC 14931 and L. reuteri ATCCPTA-4659, L. fermentum ATCC 14931, L. reuteri DSM 32273 and ATCCPTA-6475, L. fermentum ATCC 14931, L. reuteri DSM 32273 and ATCCPTA-4659, L. fermentum ATCC 14931, L. reuteri ATCC PTA-4659 and ATCCPTA-6475, or L. fermentum ATCC 14931, L. reuteri DSM 32273, ATCCPTA-6475 and ATCC PTA-4659.

In a particular embodiment, the method of FIG. 1 comprises screeningbacterial strains, other than L. reuteri DSM 32273, ATCC PTA-4659 andATCC PTA-6475, and L. fermentum ATCC 14931, for capability of producingDagK. The method also comprises selecting a bacterial strain, other thanL. reuteri DSM 32273, ATCC PTA-4659 and ATCC PTA-6475, and L. fermentumATCC 14931, which is capable of producing DagK for use in prophylaxis,inhibition and/or treatment of an allergy in a mammal.

EXAMPLES Example 1

Quantification of dagK mRNA Gene Expression by qRT-PCR

Wild type (WT) Lactobacillus reuteri ATCC PTA-6475, hdcA mutant of L.reuteri ATCC PTA-6475 (previously described in Thomas et al., 2012), WTL. reuteri ATCC PTA-4659 and WT L. reuteri DSM 17938 (deposited underthe Budapest Treaty at the DSMZ-Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (Mascheroder Weg 1 b, D-38124 Braunschweig,Germany) on Jan. 30, 2006) were grown in the MRS media overnight at 37°C. and were cultured under strict anoxic conditions N₂/CO₂ (80/20; v/v)as the gas phase. 100 μl of fresh culture was incubated into 10 ml oflactobacillus defined media 4 (LDM4). The cultures were maintained inmini bioreactors at 37° C. under strict anoxic conditions. The sampleswere collected at 3 hours, 6 hours, 24 hours and 48 hours. The bacterialpellet was obtained by treating the culture at 6000×g for 10 min at 4°C. The pellet was treated with RNase later. mRNA from the bacterialcells were extracted by Trizol separation kit. 500 ng of mRNA from eachgroup was used to convert mRNA into cDNA. The treated cDNA was diluted1:2 and was used to run qRT-PCR. The Stratagene Mx3000p (AgilentTechnologies GmbH, USA) qRT-PCR was used for amplification andfluorescent data collection. The master mix consisted of 12.5 μl PowerSYBR Green 2000 (ABI systems, USA), 0.5 μl of each primer (DAGK-L.r-F:GCGTGAGTCCATAACCGTCT (SEQ ID NO: 9) and DAGK-L.r-R: ATGGCTGCTGAAATTCCTGT(SEQ ID NO: 10), 10 μM), 1 μl of sample and adjusted with water to afinal volume of 25 μl per well. After PCR amplification, the specificityof the primers was checked by inspecting the melting curve anddetermining the size of the amplicon by agarose gel electrophoresis(1%). Relative mRNA target gene expression levels(Ratio=[(E_(target))^(dCPtarget(control-sample))]/[(E_(ref.))^(dCPref.(control-sample))])were normalized to the house keeping gene rpoB and used as a reference.Subsequently, mRNA obtained from 3 hours culture of each bacterium wereset to 1.0 and used as the calibrator to identify the relative mRNA folddifference of same bacterial strain at different time points like 6hours, 24 hours and 48 hours of L. reuteri ATCC PTA-6475, ATCC PTA-4659and DSM 17938.

FIG. 2 illustrates the results of the dagK gene expression experiments.The figure shows an increased dagK expression by both WT and hdcA mutantL. reuteri ATCC PTA-6475 together with L. reuteri ATCC PTA-4659.However, L. reuteri DSM17938 lacked dagK expression, i.e., was notcapable of producing DagK. Interestingly dagK mRNA expression wasexpressed very high during the elongation phase of the bacteria. Fromthe repetition experiments 12 hours incubation time points was selectedsince it showed similar expression like 6 hours.

Example 2

LC-MS/MS for Detecting DagK Protein in the Bacterial CultureSupernatants

According to the literatures DagK is believed to have soluble isoformsin gram positive bacteria. We hypothesized that DagK is released from L.reuteri ATCC PTA-6475 and it interacts with the host intestinalepithelial lipid signaling and suppress pro-inflammatory effects ofreleased histamine due to an allergic reaction and also promotesanti-inflammatory behavior. When we mutated the dagK gene in L. reuteriATCC PTA-6475 (mutants were created by RecT-mediated single strandrecombineering as described in van Pijkeren and Britton (2012) and isadapted to mutate the dagK gene using dagK targeted oligos) andcolonized our germ-free (GF) mice with the DagK mutant L. reuteri ATCCPTA-6475 we did not see a suppression of IL-6 and IL-la like we observedin the germ-free mice colonized with wild-type L. reuteri ATCC PTA-6475.The basal pro-inflammatory cytokine levels were significantlysuppressed. Histamine released due to an allergic reaction can activateH1R and H2R. However, H1R downstream signaling is interrupted by DagKsynthesis in L. reuteri by inhibiting lipid DAG involved in thesignaling and thereby suppress pro-inflammatory effect of histamine.This allows only H2R activation, which is known to promoteanti-inflammatory symptoms.

For dagK to show any positive effect on host immune response host-DAGlipid should be expressed. For DAG to be activated H1R signaling must beactivated. That is why when we mutated dagK in L. reuteri and colonizedthe mice we did not see pro-inflammatory cytokine suppression. This wasadditionally confirmed with PKC and PKA activation.

To further show whether DagK isoforms are secreted or not from L.reuteri we performed bacterial cell culture experiments. 100 μl ofovernight MRS grown L. reuteri ATCC PTA-6475 at 37° C. under anoxicconditions were added to 10 ml LDM4 media in the presence and absence ofDAG and left at 37° C. for 12 hours under anoxic conditions. Thebacterial cells were removed by centrifugation at 6000×g, 10 min at 4°C. 1:1 ratio of proteinase and protein kinase inhibitor was added to thesupernatant. The supernatants were filtered by 0.22 μm filter to removetraces of bacteria. Since DagK is a 10-13 kDa protein, it is necessaryto reduce the background. Therefore the supernatant was processed with50 kDa filtrate. The flow through was added to the 3 kDa filtrate andspun at 5000×g for 30 min. The concentrate on the upper phase was usedto run the LC-MS/MS after tryptic digestion. FIG. 3 illustrates theamino acid sequence of DagK protein from L. reuteri ATCC PTA-6475together with trypsin digestion or cleavage sites (Tryps). FIG. 4illustrates the amino acid sequence of DagK protein from L. reuteri ATCCPTA-6475 when DAG was present in the cell culture media.

The results of the LC-MS/MS experiment is presented in Table 1 and 2 andFIGS. 3 and 4. The sequences that match the L. reuteri DagK protein werefound in the supernatant. Accordingly, L. reuteri ATCC PTA-6475 wascapable of producing and secreting the DagK protein. Interestingly, L.reuteri secreted higher levels of DagK in the presence of DAG in cellculture media compared to its absence. However, we can detect DagK inthe presence and absence of DAG but their concentrations differ. Mediacontrol stayed negative.

TABLE 1 LC-MS/MS Results of Trypsin Treatment of Supernatant from L.reuteri ATCC PTA-6475 Peptide −10lgP Mass Length ppm m/z RT Scan #Spec A18.31 833.3813 6 −6.4 417.6953 58.33 139 3 B 14.72 971.5287 11 −62.2486.7414 63.33 432 1 C 9.09 884.4352 7 35 443.2404 72.68 1088 1 D 8.831487.774 13 −64.8 744.8461 52.15 42 1 E 7.94 996.4447 7 30.9 499.24564.98 549 1 F 5.73 2710.64 27 −79.9 678.6131 86.5 1394 1Peptides A-F in Table 1:

SEQ ID NO: 1 A EERNMR SEQ ID NO: 2 B DVAAGGVLISA SEQ ID NO: 3 C DKHQTEKSEQ ID NO: 4 D NMRYHLLAACLAI SEQ ID NO: 5 E EERNMRY SEQ ID NO: 6 FKAKDVAAGGVLISAIFSVLVGLIIFIP

TABLE 2 LC-MS/MS Results of Trypsin Treatment of Supernatant from L.reuteri ATCC PTA-6475 when DAG was present in the cell culture mediaPeptide −10lgP Mass Length ppm m/z RT Scan #Spec G 23.58 700.3755 8 1.7701.384 24.55 15840 1 H 15.34 4515.4473 39 −1.3 1129.8677 30.12 18496 1Peptides G-H in Table 2:

SEQ ID NO: 7 G DVAAGGVL SEQ ID NO: 8 HNMRYHLLAACLAIIMSILLHISAMEWLWILLAIFVVFTS

Example 3

Identification of Strains Capable of Producing DagK

The bacteria are cultivated on MRS plates for 16 h at 37° C. inanaerobic atmosphere. Bacterial colonies are collected with a sterileplastic loop and suspended in 100 μl of sterile water (PCR quality).Alternatively, DNA can be prepared from the bacterial culture using anysuitable method, see for instance Example 1.

Presence of the dagK gene is examined by PCR, e.g., by using PuReTaqReady To Go PCR beads (GE HealthCare) and the primer pair dagK_LrF(TGGACTCACGCGATAAACATCA, SEQ ID NO: 11) and dagK_LrR(ACAATCAAATCTGTAACAGCTTCG, SEQ ID NO: 12), 0.4 mM of each. Bacterialsuspension or DNA preparation (0.5 μl) is added to the PCR mix and thePCR reaction is performed by running the program 95° C., 5 min; 30× (95°C., 30 s; 58° C., 30 s; 72° C., 30 s); 72°, 10 min. The PCR products areseparated and visualized by using standard agarose gel electrophoresisand the sequence is determined by standard Sanger sequencing using theforward primer (dagK_LrF) used for the PCR.

Example 4

Analysis of Lactobacillus reuteri DSM 32273 Capable of Producing DagK

Lactobacillus reuteri DSM 32273 bacteria were grown over night in MRSbroth at 37° C. The bacterial suspensions were centrifuged at 3500 rpmfor 5 min and 1 μl of the pellet was suspended in 100 μl of PBS.

The PCR analysis of dagK gene in L. reuteri DSM 32273 was done asdescribed in Example 3. The results showed that L. reuteri DSM 32273 waspositive for the gene encoding histidine decarboxylase and the dagKgene, see Table 3. The bacterial strains L. reuteri ATCC PTA-6475 andDSM 17938 were included as controls.

TABLE 3 Results from the PCR Analysis Showing the Species, Strain andHost Origin of the Tested bacteria. Species Strain Host origin Presenceof dagK gene L. reuteri ATCC PTA-6475 Human + DSM 17938 Human − DSM32273 Human +

Example 5

Manufacture of a Probiotic Product for Use in Pollen Allergy

In this example, a probiotic product for use in pollen allergy ismanufactured. The strain L. fermentum ATCC 14931 is selected based onits capability to produce DagK as analyzed from the published genomesequence. The L. fermentum strain is grown and lyophilized, usingstandard methods for growing Lactobacillus in the industry. The productis an oil-based formulation made for good stability and shelf life. Theunique feature of production process is the step of drying the oil byplacing it under vacuum to remove most of the water in the oil and toincrease the stability in the formulation. The oil used in the inventionherein is a pure edible vegetable oil, preferably sunflower oil andmedium-chain triglyceride.

Mixing of Ingredients.

1. Mix the medium-chain triglyceride, for example, Akomed R (KarlshamnsAB, Karlshamn Sweden), and sunflower oil, for example, Akosun(Karlshamns AB, Karlshamn Sweden) with silicon dioxide, Cab-o-sil M5P,M5P, Cabot) in a Bolz mixing machine/tank (Alfred BOLZ Apparatebau GmbH,Wangen im Allgäu, Germany).

2. Homogenization. A Sine pump and dispax (Sine Pump, Arvada, Colo.) areconnected to the Bolz mixer and the mixture is homogenized.

3. Vacuum-drying. The mixture is dried under 10 mBar vacuum in the Bolztank, for 12 hours.

4. Adding Lactobacillus fermentum. About 20 kg of dried oil mixture ismoved to a 50 liter stainless steel vessel. L. fermentum powder,preferably freeze-dried; the amount of L. fermentum used would varydepending on the amount wanted in the oil, but one example would be toadd 0.2 kg of culture having 10¹¹ CFU per g, is added. It is mixedslowly until homogenous.

5. Mixing. The premix with L. fermentum is brought back to the Bolzmixer.

6. Discharging. The suspension is discharged to a 200 liter glassvessel, and covered with nitrogen.

The suspension is held in the vessel until filling in spray bottles tobe used for nasal administration to a human for the prevention ortreatment of pollen allergy.

Example 6

Manufacture of a Probiotic Product for Use in Food Allergy

In this example, Lactobacillus reuteri DSM 32273 is selected based onits capability to produce DagK in order to add the strain to a tabletfor use in the prevention, inhibition and/or treatment of food allergyin humans. The L. reuteri strain is grown and lyophilized, usingstandard methods for growing Lactobacillus in the industry.

The following steps illustrate an example of a manufacturing process fortablets containing the selected bacterial strain, including glucoseencapsulation. It is understood that excipients, fillers, flavors,encapsulators, lubricants, anticaking agents, sweeteners and othercomponents of tablet products as are known in the art, may be usedwithout affecting the efficacy of the product:

1. Melting. Melt SOFTISAN™ 154 (SASOL GMBH, Bad Homburg, Germany) in avessel and heat it to 70° C. to assure complete disruption of thecrystalline structure. Then cool it down to 52-55° C. (just above itshardening point).

2. Granulation. Transfer Lactobacillus reuteri freeze-dried powder to aDiosna high-shear mixer/granulator, or equivalent. Add slowly duringapproximately 1 minute the melted SOFTISAN™ 154 to the L. reuteripowder. Use chopper during the addition.

3. Wet-sieving. Immediately after the granulation, pass the granulesthrough a 1-mm sieving net by using a Tornado mill. The sieved granulateis packed in alupouches, made out of PVC-coated aluminum foil, sealedwith a heat sealer to form a pouch, together with desiccant pouch, andstored refrigerated until mixing. The granulated batch is divided fortwo tablet batches.

4. Add encapsulated D-Glucose (G8270, >99.5% glucose, Sigma),encapsulated using standard microencapsulating methods as known in theart. The amount of sugar is dependent on the total CFU of the addedpowder of dry L. reuteri, a standard level can be 1 gram of sugar pertotal CFU of 10⁸ of bacteria but this could also be varied down to 0.1gram or 0.01 gram up to 10 gram even up to 100 gram of sugar.

5. Mixing. Mix all the ingredients in a mixer, to a homogenous blend.

6. Compression. Transfer the final blend to the hopper of a rotarytablet press and compress tablets with a total weight of 765 mg, in aKilian compressor.

7. Bulk packaging. The tablets are packed in alu-bags together with adrying pouch of molecular sieve. The alu-pouch is put in a plasticbucket and stored in a cool place at least one week, before finalpackage. The use of SOFTISAN™, a hydrogenated palm oil, enables theLactobacillus cells to be encapsulated in fat and environmentallyprotected.

As stated above, the product of the embodiments may be in forms otherthan tablet, and standard methods of preparing the underling underlyingproduct as are known in the art are beneficially used to prepare theproduct of the invention including the selected L. reuteri culture.

Example 7

Mouse Enteroid Experiment to Explore Mammalian Intestinal Epithelial DAGand pPKC Signaling

Mouse enteroids (contains only intestinal epithelial layer) were derivedfrom 10 week-old germ free (GF) BALB/c mouse. The enteroids were grownto form a monolayer and were induced with LDM4 media (control),wild-type L. reuteri PTA-6475 conditioned media (50 to 3 kDa cut off),diacylglycerol kinase (DGK) inhibitor (2 μM) only or DGK inhibitor (2μM) with wild-type L. reuteri PTA-6475 conditioned media (CM; 50 to 3kDa cut off). DGK inhibitor (R59-022;6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo-[3,2-a]pyrimidine-5-one)was added 2 hours prior to the addition of conditioned media onenteroids to prevent the mammalian DGK activation and after 2 hours theenteroids were washed and treated with conditioned media or only LDM4media with or without DGK inhibitor respectively. The proteins werecollected after 45 min of incubation to perform Western blot.

The enteroids treated with a DGK inhibitor yielded increased PKCphosphorylation in the presence of wild-type (WT) L. reuteri PTA-6475conditioned media, while enteroids lacking DGK inhibitor in the presenceof wild-type L. reuteri PTA-6475 conditioned media did not yieldevidence of increased PKC phosphorylation, see FIGS. 5a and 5 b.

The embodiments described above are to be understood as a fewillustrative examples of the present invention. It will be understood bythose skilled in the art that various modifications, combinations andchanges may be made to the embodiments without departing from the scopeof the present invention. In particular, different part solutions in thedifferent embodiments can be combined in other configurations, wheretechnically possible. The scope of the present invention is, however,defined by the appended claims.

REFERENCES

-   Thomas, et al. (2012). Histamine derived from probiotic    Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK    signaling. PLoS One 7(2): e31951.-   van Pijkeren and Britton. (2012). High efficiency recombineering in    lactic acid bacteria. Nucleic Acids Research 40(10): e76.-   Lee, et al. (2004). Dietary intake of various lactic acid bacteria    suppresses type 2 helper T cell production in antigen-primed mice    splenocyte. J. Microbiol. Biotechnol. 14(1): 167-170.

The invention claimed is:
 1. A method of treating or reducing the risk of developing a food allergy in a mammal, comprising administering to a mammal in need thereof a diacylglycerol kinase (DagK) producing lactic acid bacterial strain that is capable of releasing DagK extracellularly, wherein the DagK producing lactic acid bacterial strain is not L. reuteri strain ATCC PTA-6475.
 2. The method of claim 1, wherein the lactic acid bacterial strain is a Lactobacillus reuteri bacterial strain.
 3. The method of claim 1, wherein the lactic acid bacterial strain is L. reuteri strain DSM
 32273. 4. The method of claim 1, wherein the lactic acid bacterial strain is L. reuteri strain ATCC PTA-4659.
 5. The method of claim 1, further comprising administering an H1-antihistamine.
 6. The method of claim 5, wherein the H1 antihistamine is selected from a group consisting of cetirizine, ebastine, fexofenadine, loratadine and desloratadine. 